Studies from this laboratory have demonstrated that diphenylhydantoin (DPH) and calcium ions had antagonistic actions on the level of endogenous phosphorylation of two specific proteins from rat and human brain preparations, designated proteins DPH-L and DPH-M. An hypothesis was proposed from these results that some of the effects of DPH on neuronal tissue and seizure discharge might be mediated by the action of DPH on protein phosphorylation, and that phosphoproteins may play a role in mediating the antagonistic actions of DPH and calcium on the release of neurotransmitter during posttetanic potentiation. The proposed investigation will systematically examine this hypothesis and delineate the role of phosphoproteins in the action of anticonvulsants and seizure discharge. The initial phase of this effort will be directed at characterizing the effects of DPH on protein phosphorylation (mechanism of action, subcellular distribution, identification of substrate), and determining the effects of other representative anticonvulsants and select neuropharmacologic agents on the phosphorylation of proteins DPH-L and DPH-M. The technique of subcellular fractionation, SDS-acrylamide gel electrophoresis, and autoradiography will be utilized to isolate and quantify the incorporation of (32P) phosphate into specific brain proteins. The second phase of this investigation will be directed at correlating the effect of DPH, other anticonvulsants, and specific neuropharmacologic agents on the level of phosphorylation of specific brain proteins with their known and experimentally determined actions on the release of neurotransmitter from nerve terminals, and their effects on seizure thresholds in several experimental animal models. This research should provide information of potential practical significance in our knowledge of the molecular action of anticonvulsants and will elucidate the possible role of phosphoproteins in neurotransmitter release and seizure discharge.